CN116286762A - Glutamate decarboxylase and application thereof - Google Patents
Glutamate decarboxylase and application thereof Download PDFInfo
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- CN116286762A CN116286762A CN202310148159.7A CN202310148159A CN116286762A CN 116286762 A CN116286762 A CN 116286762A CN 202310148159 A CN202310148159 A CN 202310148159A CN 116286762 A CN116286762 A CN 116286762A
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- decarboxylase
- glutamate decarboxylase
- glutamic acid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Abstract
The invention provides glutamate decarboxylase and application thereof, and relates to the technical field of biology. The amino acid sequence of the glutamic acid decarboxylase provided by the invention is shown as SEQ ID NO. 1. The glutamic acid decarboxylase is obtained by further digging on the basis of the existing glutamic acid decarboxylase, and the invention researches show that the glutamic acid decarboxylase provided by the invention has good pH stability and thermal stability, higher catalytic activity and wider pH application range than those before improvement, and has good prospect of industrialized production of gamma-aminobutyric acid.
Description
Technical Field
The invention relates to the technical field of biology, in particular to glutamate decarboxylase and application thereof.
Background
Gamma-aminobutyric acid (GABA), also known as 4-aminobutyric acid, is a four-carbon non-protein amino acid widely existing in animals, plants and microorganisms, and has physiological functions of regulating hormone secretion, reducing blood pressure, controlling asthma, tranquilizing and allaying excitement, enhancing memory, treating nervous system diseases, resisting aging and the like. In addition, GABA is also an important intermediate for synthesizing nylon-4 and 2-pyrrolidone, and has wide application prospect in the industries of food, medicine, chemical industry and the like.
As shown in FIG. 1, glutamate decarboxylase (GAD, EC 4.1.1.15) is capable of catalyzing the irreversible alpha-decarboxylation of glutamic acid (L-Glu) or sodium glutamate (L-MSG) to GABA in the presence of the cofactor pyridoxal phosphate (PLP). GAD is widely existed in microorganisms, and two main defects of the GAD in application are poor thermal stability and the GAD can only play a role under an acidic condition, and the poor thermal stability leads to extremely easy inactivation of enzymes in large-scale catalytic production of GABA, continuous acid or alkali addition, equipment corrosion, and extremely increased production cost. In order to solve these problems, in recent years, researchers have made much effort in developing biocatalysts with improved thermal stability and/or an effectively widened pH applicability.
Based on the fact that there is no absolute correspondence between the primary structure of the protein and the function of protein expression, the high sequence consistency does not represent similar catalytic properties, and if the key catalytic sites are mutated, the catalytic properties are greatly different.
In view of this, the present invention has been made.
Disclosure of Invention
It is a first object of the present invention to provide a glutamate decarboxylase which solves at least one of the above problems.
A second object of the present invention is to provide the use of the above glutamic acid decarboxylase in the catalytic synthesis of gamma-aminobutyric acid.
The third object of the present invention is to provide a product for the catalytic synthesis of gamma-aminobutyric acid.
A fourth object of the present invention is to provide a gene.
A fifth object of the present invention is to provide a recombinant plasmid.
The sixth object of the present invention is to provide a genetically engineered bacterium.
The seventh object of the present invention is to provide a method for producing glutamic acid decarboxylase.
In a first aspect, the present invention provides a glutamate decarboxylase having the amino acid sequence shown in SEQ ID NO. 1.
In a second aspect, the present invention provides the use of the glutamate decarboxylase described above in the catalytic synthesis of gamma-aminobutyric acid.
In a third aspect, the present invention provides a product for the catalytic synthesis of gamma-aminobutyric acid, namely said glutamate decarboxylase;
in a fourth aspect, the present invention provides a gene encoding the glutamate decarboxylase.
As a further technical scheme, the nucleic acid sequence of the gene is shown as SEQ ID NO. 2.
In a fifth aspect, the present invention provides a recombinant plasmid comprising a vector and said gene.
As a further embodiment, the vector comprises pET-28a (+).
In a sixth aspect, the present invention provides a genetically engineered bacterium, which contains the recombinant plasmid.
As a further technical scheme, the genetically engineered bacterium is escherichia coli.
In a seventh aspect, the invention provides a preparation method of glutamate decarboxylase, which comprises the steps of fermenting the genetically engineered bacterium, and separating and purifying to obtain the glutamate decarboxylase.
Compared with the prior art, the invention has the following beneficial effects:
the glutamic acid decarboxylase provided by the invention is obtained by further digging on the basis of the existing glutamic acid decarboxylase, and the invention researches show that the glutamic acid decarboxylase provided by the invention has good pH stability and thermal stability, higher catalytic activity and wider pH application range than those before improvement, and has good prospect of industrialized production of gamma-aminobutyric acid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a reaction equation for glutamate decarboxylase catalyzed gamma-aminobutyric acid;
FIG. 2 is an SDS-PAGE electrophoresis of glutamate decarboxylase;
FIG. 3 is a graph showing the effect of pH on glutamate decarboxylase activity;
FIG. 4 is a graph showing the effect of temperature on glutamate decarboxylase activity;
FIG. 5 shows the pH stability of glutamate decarboxylase;
FIG. 6 shows the thermostability of glutamate decarboxylase;
FIG. 7 shows the progress of a reaction for producing gamma-aminobutyric acid by the catalysis of glutamate decarboxylase.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but it will be understood by those skilled in the art that the following embodiments and examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not specified, and the process is carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In a first aspect, the present invention provides a glutamate decarboxylase, the amino acid sequence of which is shown in SEQ ID NO. 1:
MGTLVSKDDIREKLYDSTYAARDLARPLPKYAFPEIEHDPKAIMQVVEDELFLDGNPRQNLATFCQTWEEEEVHKIMDFSMDRNMIDKSEYPQTAELEIRCAHMLADLWNAPDAEDTIGTSTTGSSEACMLGGMAAKWRWRAKRQAEGKPIDKPNLVCGPVQVCWEKFCRYWDIEKREVEMTPDRLQMDPASMLELVDENTIAVVPTFGVTYTGHYELVKPLSDALDKLQEEKGLDIDIHVDAASGGFLGPFCAPEIEWDFRLPRVKSISTSGHKYGLAPLGVGWIVWRDKAELPDDLVFSVPYLGGSVGTFAINFSRPAGQIVSQYYLFNRLGRDGYRRVQDASYQVAQFLSSRLEKLGPFDFINTGDPKAGIPAVCFRIRDGADLPYTLYDLSAKLLERGWQVPAFNLSGKASDITIMRVMVRQGVSMDMAQLLVEDIERAIAHFDAHPVTTPLTEEERGSYSHG(SEQ ID NO.1)。
the glutamic acid decarboxylase provided by the invention is obtained by further digging by taking the existing glutamic acid decarboxylase as a template, the sequence homology of the enzyme and GadB (gamma-associated protein) from a template Escherichia coli K-12 source is 59.27%, the recombinant glutamic acid decarboxylase has 466 amino acids in total, and the molecular weight is about 53.5kDa. The invention researches show that the glutamic acid decarboxylase provided by the invention has good pH stability and thermal stability, has higher catalytic activity and wider pH application range than the glutamic acid decarboxylase before improvement, and has good prospect of industrialized production of gamma-aminobutyric acid.
In a second aspect, the present invention provides the use of the glutamate decarboxylase described above in the catalytic synthesis of gamma-aminobutyric acid.
The glutamic acid decarboxylase provided by the invention has good stability, higher catalytic activity and wider pH application range, so that the glutamic acid decarboxylase provided by the invention is applied to the catalytic synthesis of gamma-aminobutyric acid, and is beneficial to reducing the production cost.
In a third aspect, the present invention provides a product for the catalytic synthesis of gamma-aminobutyric acid, said product comprising said glutamate decarboxylase;
in addition, the glutamic acid decarboxylase can be immobilized on an enzyme carrier by an immobilization method, so that an immobilized enzyme product is prepared.
In the present invention, the enzyme carrier is not particularly limited, and a carrier capable of immobilizing the enzyme, which is well known to those skilled in the art, may be used, and the glutamate decarboxylase may be immobilized on the enzyme carrier to thereby reuse the glutamate decarboxylase.
In a fourth aspect, the present invention provides a gene encoding the glutamate decarboxylase.
As a further technical scheme, the nucleic acid sequence of the gene is shown as SEQ ID NO. 2:
ATGACCCTGGTGAGCAAAGATGATATTCGCGAAAAACTGTATGATAGCACCTATGCGGCGCGCGATCTGGCGCGCCCGCTGCCGAAATATGCGTTTCCGGAAATTGAACATGATCCGAAGGCGATTATGCAAGTGGTGGAAGATGAACTGTTTCTGGATGGCAACCCGCGTCAGAACCTGGCGACCTTTTGTCAGACCTGGGAAGAGGAAGAAGTGCATAAAATTATGGATTTTAGCATGGATCGCAACATGATTGATAAAAGCGAATATCCGCAGACCGCGGAACTGGAAATTCGCTGCGCGCACATGCTGGCGGATCTGTGGAACGCGCCGGATGCGGAAGATACCATTGGCACGAGCACCACCGGCAGCAGCGAAGCGTGCATGCTGGGCGGCATGGCGGCGAAATGGCGCTGGCGCGCGAAACGCCAAGCGGAAGGCAAACCGATTGATAAACCGAACCTGGTGTGCGGCCCGGTGCAAGTGTGCTGGGAAAAATTTTGCCGCTATTGGGATATTGAAAAACGCGAAGTGGAAATGACCCCGGATCGCCTGCAGATGGATCCGGCGAGCATGCTGGAACTGGTGGATGAAAACACCATTGCGGTGGTGCCGACCTTTGGCGTGACCTATACCGGCCATTATGAACTGGTGAAACCGCTGAGCGATGCGCTGGATAAACTGCAAGAAGAAAAAGGCCTGGATATTGATATTCATGTGGATGCGGCGAGCGGCGGCTTTCTGGGCCCGTTTTGCGCGCCGGAAATTGAGTGGGATTTTCGCCTGCCGCGCGTGAAAAGCATTAGCACGAGCGGCCATAAATATGGCCTGGCGCCGCTGGGCGTGGGCTGGATTGTGTGGCGCGATAAAGCGGAACTGCCGGATGATCTGGTGTTTAGCGTGCCGTATCTGGGCGGCAGCGTGGGCACCTTTGCGATTAACTTTAGCCGCCCGGCGGGTCAGATTGTGAGTCAGTATTATCTGTTTAACCGCCTGGGCCGCGATGGCTATCGCCGCGTGCAAGATGCGAGCTATCAAGTGGCGCAGTTTCTGAGCAGCCGCCTGGAAAAACTGGGCCCATTCGATTTTATTAACACCGGCGATCCAAAAGCCGGCATCCCGGCGGTGTGCTTTCGCATTCGCGATGGCGCGGATCTGCCGTATACCCTGTATGATCTGAGCGCGAAACTGCTGGAACGCGGCTGGCAAGTGCCGGCGTTTAACCTGAGCGGCAAAGCGAGCGATATTACCATTATGCGCGTGATGGTGCGCCAAGGCGTGAGCATGGATATGGCGCAGCTGCTGGTGGAAGATATTGAACGCGCGATTGCGCATTTTGATGCGCATCCGGTGACCACCCCGCTGACCGAAGAGGAACGCGGCAGCTATAGCCATGGCTAA(SEQ ID NO.2)。
in a fifth aspect, the present invention provides a recombinant plasmid comprising a vector and said gene. The recombinant plasmid can realize the expression of glutamate decarboxylase.
As a further alternative, the vector may include, but is not limited to, pET-28a (+), or may be other vectors known to those skilled in the art.
In a sixth aspect, the present invention provides a genetically engineered bacterium, which contains the recombinant plasmid.
The genetically engineered bacterium can express glutamate decarboxylase.
As a further technical scheme, the genetically engineered bacterium is escherichia coli.
In a seventh aspect, the invention provides a preparation method of glutamate decarboxylase, which comprises the steps of fermenting the genetically engineered bacterium, and separating and purifying to obtain the glutamate decarboxylase.
The method for separating and purifying the glutamic acid decarboxylase in the fermentation product is not particularly limited, and methods which are well known to those skilled in the art and can be used for separating the fermentation product may be adopted.
The preparation method provided by the invention is simple and convenient, and can be used for mass production of glutamate decarboxylase.
The invention is further illustrated by the following specific examples and comparative examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and should not be construed as limiting the invention in any way.
The preparation raw materials selected in the following examples are as follows:
pET-28a (+) vector: from Novagen corporation;
coli BL21 (DE 3): from Beijing full gold biotechnology Co., ltd;
LB medium: 10g pancreatic protein jelly (Tryptone) per liter, 5g Yeast extract (Yeast extract), 5g NaCl,1ml 1mol/L NaOH to adjust pH to 7.4, and sterilizing with deionized water to 1L under high pressure steam for 20min;
TB medium: 12g Tryptone (Tryptone), 24g Yeast extract (Yeast extract), 4mL of 87% glycerol, 100mL of phosphate buffer solution (pH 6.5) per liter, and steam sterilized with deionized water to a volume of 1L for 20min under high pressure.
EXAMPLE 1 Gene dig of glutamate decarboxylase
This example relates to the use of databases for gene mining to obtain Marinibacterium profundimaris-derived glutamate decarboxylase genes (SEQ ID NO.2, amino acid sequence of which is SEQ ID NO. 1). .
EXAMPLE 2 preparation of recombinant bacteria and fermentation expression
The present example relates to the construction of recombinant plasmids, and the heterologous expression of glutamate decarboxylase by recombinant strains is constructed by taking E.coli as an example, and the recombinant strains are subjected to fermentation culture and induced expression.
a1. Constructing a recombinant plasmid, connecting the N-terminal of an amino acid sequence shown in SEQ ID NO.2 with a 6 xHis tag, and then connecting the recombinant plasmid to an expression vector pET-28a (+) to obtain a recombinant plasmid pET-28a (+) -6 xHis-GAD containing a glutamate decarboxylase gene, and verifying the sequencing to be correct;
a2. construction of a recombinant strain, namely, transforming a recombinant plasmid pET-28a (+) -6 xHis-GAD into escherichia coli BL21 (DE 3) to obtain a recombinant strain E.coli BL21 (DE 3)/pET-28 a (+) -6 xHis-GAD;
a3. culturing seed liquid: inoculating recombinant bacteria into 10mL of LB liquid medium, wherein the medium contains 50 mug/mL kanamycin with final concentration, and culturing for 16h at 37 ℃ under shaking at 180 rpm;
a4. and (3) performing expansion culture: inoculating the seed solution into 50mL of TB culture medium (the inoculum size is 1%), wherein the culture medium contains kanamycin with the final concentration of 50 mug/mL, carrying out shaking culture at 37 ℃ and 180rpm until the bacterial liquid OD600nm is 0.6-0.8, adding IPTG with the final concentration of 0.5 mu M, continuing shaking culture at 20 ℃ and 180rpm for 20-24h, centrifuging at 600 rcf for 5min, carrying out buffer washing twice, and collecting bacterial cells;
a5. and (3) thallus crushing: after being resuspended in 50mL of disodium hydrogen phosphate-citric acid buffer (20 mM pH 7.0), the mixture was crushed by a high-pressure homogenizer, centrifuged at 12000rpm for 20min, and the supernatant was collected to obtain a crude enzyme solution of glutamate decarboxylase, which was then separated and purified by an AKTA protein purifier to obtain a pure enzyme (designated as Mp-GAD).
EXAMPLE 3 SDS-PAGE electrophoretic analysis of glutamate decarboxylase
This example relates to SDS-PAGE electrophoretic analysis of recombinant glutamate decarboxylase comprising the steps of:
b1. preparing separation gel and concentrated gel, placing the gel plate in an electrophoresis tank, and pouring 440mL of 1 XTris-Glycine buffer solution;
b2. mixing 80 μl of protein solution with 20 μl of 2×loading buffer and 5 μl of beta-mercaptoethanol, heating at 95deg.C for 5min, and loading 10 μl;
b3. after the sample loading is finished, a power supply is switched on, electrophoresis is carried out at 105V until the bromophenol blue strip migrates to the joint of the concentrated glue and the separating glue, the constant voltage is switched to 155V until the bromophenol blue strip completely runs out of the glass plate, the electrophoresis is stopped, and the power supply is switched off;
b4. the concentrated gel was cut with a blade and the gel was stained in coomassie brilliant blue R-250 gel stain for 30min. The gel was then transferred to a destaining solution for destaining until a clear protein band was seen, as shown in FIG. 2, there was a distinct protein band around 50kDa, demonstrating successful expression of glutamate decarboxylase.
Example 4 determination of the enzymatic Properties of glutamate decarboxylase
This example relates to the enzymatic properties of glutamate decarboxylase:
method for measuring enzyme activity: based on Berchelot reaction, an appropriate amount of enzyme solution was added to a citric acid-disodium hydrogen phosphate buffer (50 mmol/L, pH 4.5) containing a substrate L-MSG at a final concentration of 24mmol/L and a cofactor PLP at a final concentration of 24. Mu. Mol/L, and the total reaction system was 250. Mu.L. After 5min of reaction, 375. Mu.L of boric acid-sodium tetraborate buffer (0.2 mol, pH 9.0) was added, mixed well, inactivated at 100℃for 5min, then 250. Mu.L of redistilled phenol (6% (w/v)) and 50. Mu.L of sodium hypochlorite (6-14% active chlorine) were sequentially added, reacted at 100℃for 5min, immediately subjected to ice-water bath, after 5min, 200. Mu.L of the reaction solution was taken in a 96-well plate, the absorbance at lambda 630 was measured by using a microplate reader, and the GABA content was calculated.
The protein content determination method comprises the following steps: the protein content in the system was determined based on the Bradford method. Taking 1mL of enzyme solution with proper concentration, adding 5mL of coomassie brilliant blue G-250, reacting for 5min, measuring the absorbance value of the reaction solution at lambda 595 by using an ultraviolet spectrophotometer, and calculating the protein content in the enzyme solution.
A glutamate decarboxylase enzyme solution (designated Ec-GAD) was prepared by the method provided in example 2, using the amino acid sequence of Escherichia coli K-12-derived glutamate decarboxylase (NCBI accession number: NP-416010.1) as a template. The specific enzyme activity of Ec-GAD was measured by the method for measuring enzyme activity described above, and the results are shown in Table 1.
TABLE 1 comparison of GADs Activity
c1. Determination of optimum pH: and (3) taking a proper amount of GAD enzyme liquid, respectively adding the GAD enzyme liquid into citric acid-disodium hydrogen phosphate buffer solution (50 mmol/L, pH is 3.0-7.0), incubating for 5min at 37 ℃, and measuring the enzyme activity of the GAD enzyme liquid in different pH buffers, wherein the enzyme activity under the optimal pH condition is used as a control (100%). As shown in FIG. 3, the optimum pH of the enzyme was 4.5.
c2. Determination of the optimum temperature: taking a proper amount of GAD enzyme solution, respectively incubating for 5min at 20-70 ℃, and measuring the enzyme activities at different temperatures at the respective optimal pH values, wherein the enzyme activities at the optimal temperatures are used as a control (100%). As shown in FIG. 4, the optimum temperature of the enzyme was 40 ℃.
Measurement of ph stability: the GAD enzyme solutions were incubated in citric acid-disodium hydrogen phosphate buffer (50 mmol/L) at pH4.0 and pH7.0, respectively, at room temperature for a certain period of time, and the remaining enzyme activities were measured by sampling at regular intervals, with the enzyme activities incubated for 0h as a control (100%). The pH stability of the template Ec-GAD is shown as A in FIG. 5, the pH stability of the Mp-GAD is shown as B in FIG. 5, after incubation in buffer solution at pH4.0 for 20h, the residual enzyme activity of the Mp-GAD is 31.07% of the initial enzyme activity, and the residual enzyme activity of the template Ec-GAD is 28.12% of the initial enzyme activity; after incubation in buffer solution at pH7.0 for 20h, the Mp-GAD residual enzyme activity was 49.35% of the initial enzyme activity, and the template Ec-GAD residual enzyme activity was 34.92% of the initial enzyme activity. In conclusion, the stability of Mp-GAD at pH4.0 and pH7.0 was improved to some extent as compared with that of the template Ec-GAD.
c4. Determination of thermal stability: the GAD enzyme solutions were incubated at 37℃for 10 hours, and the remaining enzyme activities were sampled at regular intervals, and the enzyme activities incubated at 37℃for 0 hour were used as controls (100%). As shown in FIG. 6, after incubation at 37℃for 10 hours, the remaining enzyme activity of MP-GAD was 50.75% of the initial enzyme activity, and MP-GAD had better thermostability than the template Ec-GAD (36.84%).
EXAMPLE 5 catalytic synthesis of gamma-aminobutyric acid by glutamate decarboxylase
This example relates to experiments for the catalytic synthesis of gamma-aminobutyric acid by glutamate decarboxylase.
Reaction system (10 mL): comprises a final concentration of 24mmol/L of substrate L-MSG, 24. Mu. Mol/L of cofactor PLP, and a buffer solution of citric acid and disodium hydrogen phosphate (50 mmol/L, pH 4.5) and 1.961mg of crude enzyme solution. The reaction was carried out in a shaker at 37℃and 180rpm for 1 hour, the sample was boiled and inactivated at 100℃for 10 minutes, and then the GABA yield was measured by high performance liquid chromatography after pre-column derivatization. As shown in FIG. 7, the GABA yield increased gradually with time, and at 40min of reaction, the GABA yield could reach 97.58%, and as the reaction time continued to be prolonged, the GABA yield was not increased any more. Experimental results show that the glutamate decarboxylase has good catalytic effect on catalyzing L-MSG to generate GABA through alpha-decarboxylation reaction.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. The glutamic acid decarboxylase is characterized in that the amino acid sequence of the glutamic acid decarboxylase is shown as SEQ ID NO. 1.
2. Use of the glutamate decarboxylase of claim 1 for the catalytic synthesis of gamma-aminobutyric acid.
3. A product for the catalytic synthesis of gamma-aminobutyric acid, comprising the glutamate decarboxylase of claim 1.
4. A gene encoding the glutamate decarboxylase of claim 1.
5. The gene according to claim 4, wherein the nucleic acid sequence of the gene is shown in SEQ ID NO. 2.
6. A recombinant plasmid comprising a vector and the gene of claim 4 or 5.
7. The recombinant plasmid of claim 6, wherein the vector comprises pET-28a (+).
8. A genetically engineered bacterium comprising the recombinant plasmid of claim 6 or 7.
9. The genetically engineered bacterium of claim 8, wherein the genetically engineered bacterium is e.
10. The method for producing glutamic acid decarboxylase according to claim 1, wherein the glutamic acid decarboxylase is produced by fermenting the genetically engineered bacterium according to claim 8 or 9 and separating and purifying the bacterium.
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| JP2005124488A (en) * | 2003-10-24 | 2005-05-19 | National Institute Of Advanced Industrial & Technology | HEAT-RESISTANT GLUTAMIC ACID DECARBOXYLASE, DNA ENCODING THE SAME, METHOD FOR PRODUCING THE ENZYME, AND METHOD FOR PRODUCING SUBSTANCE(gamma-AMINOBUTYRIC ACID OR THE LIKE) USING THE ENZYME |
| KR20170099510A (en) * | 2016-02-24 | 2017-09-01 | 충북대학교 산학협력단 | Highly active GABA-producing glutamate decarboxylase from Bacteroides sp. and use thereof |
| CN110760533A (en) * | 2019-12-05 | 2020-02-07 | 南阳师范学院 | Gene for coding glutamate decarboxylase, recombinant engineering bacterium and application thereof |
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| JP2005124488A (en) * | 2003-10-24 | 2005-05-19 | National Institute Of Advanced Industrial & Technology | HEAT-RESISTANT GLUTAMIC ACID DECARBOXYLASE, DNA ENCODING THE SAME, METHOD FOR PRODUCING THE ENZYME, AND METHOD FOR PRODUCING SUBSTANCE(gamma-AMINOBUTYRIC ACID OR THE LIKE) USING THE ENZYME |
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